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Single cell nucleus extraction method suitable for frozen tissue

An extraction method and single-cell nucleus technology, which can be used in biochemical equipment and methods, recombinant DNA technology, and microbial assay/inspection. ratio, the operation process is simple, and the effect of strong practicability

Inactive Publication Date: 2021-08-24
SINGLERON NANJING BIOTECHNOLOGIES LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The single cell nucleus extraction method provided by the present invention minimizes the degradation of RNA, and solves the problems of long operation time, cumbersome process and high cost in the existing single cell nucleus extraction from frozen tissue

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] In this example, 10 mL of a NEBCS nuclear separation solution was prepared, and 1 mL of 3.2 M sucrose, 100 μL of 500 mM CaCl 2 , 100 μL 300 mM Mg(Ac) 2 , 100 μL 10 mM EDTA, 100 μL 2 M Tris-HCl, 80 μL 10% Triton X-100, 800 μL 100 mM DTT and 50 μL 40 U / μL RNase inhibitor in a 15 mL EP tube, filled with nuclease-free water Make up to 10 mL to get the NEBCS nuclear separation solution.

Embodiment 2

[0026] In this example, 10 mL of a NEBCS nuclear separation solution was prepared, and 1 mL of 3.2 M sucrose, 100 μL of 500 mM CaCl 2 , 100 μL 300 mM Mg(Ac) 2 , 100 μL 10 mM EDTA, 100 μL 2 M Tris-HCl, 50 μL 10% Triton X-100, 500 μL 100 mM DTT and 50 μL 40 U / μL RNase inhibitor in a 15 mLEP tube, filled up with nuclease-free water to 10 mL to obtain NEBCS nuclear separation solution.

Embodiment 3

[0028] In this example, the NEBCS nuclear separation solution configured in Example 1 was used to extract a single nucleus from human cryopreserved bone cancer tissue. The specific extraction steps are as follows:

[0029] (1) Take 50 mg of frozen human bone cancer tissue into a 1.5 mL sterile centrifuge tube, place it in an ice bath, add 100 μL of 4°C pre-cooled NEBCS nuclear separation solution, and immediately cut the bone cancer tissue into small pieces with surgical scissors. Add 1 mL of NEBCS nuclear separation solution to the mince, incubate on ice for 5-10 min, and mix by inverting every 2 min;

[0030] (2) After the incubation, use a 50 um filter to filter into a 15 mL centrifuge tube, wash the filter with 4 mL of pre-cooled PBS-RI buffer; centrifuge at 100-300 rcf for 1-3 minutes at 4°C, and draw 4.5 mL Transfer the supernatant to a new 15mL centrifuge tube; centrifuge at 500 rcf for 5 min, fully remove the supernatant, add 50 μL pre-cooled PBS-RI buffer to resuspend...

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Abstract

The invention relates to the technical field of biology, in particular to a single cell nucleus extraction method suitable for frozen tissue. According to the method disclosed by the invention, the single cell nucleus is successfully separated and extracted from the frozen tissue in a relatively shorter time by using the NEBCS nucleus separation liquid, and the more pure single cell nucleus is obtained through differential centrifugation. The proportion of cell nucleus RNA degradation is greatly reduced, so that protein coding RNA with a higher proportion can be obtained at the single cell level, and the proportion of mitochondrial genes is effectively reduced. The mononuclear extraction method provided by the invention is suitable for various types of frozen tissues, and further degradation by an RNA method is effectively prevented. The operation process is simple, the extraction process of the cell nucleus can be completed by using common laboratory instruments such as a centrifugal machine and a microscope, and the whole process only needs 30-60 minutes. Special instruments and devices are not needed, so that the extraction cost is effectively reduced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a single cell nucleus extraction method suitable for frozen tissues. Background technique [0002] Single-cell whole-transcriptome sequencing technology is a new technology for amplifying and sequencing whole-transcriptome at the single-cell level. Its principle is to amplify a small amount of whole-transcriptome RNA from a single cell isolated and perform high-throughput sequencing. It is a tool for studying cell types, dynamics, and functional processes in complex tissues. Compared with traditional transcriptome sequencing, single-cell sequencing not only measures gene expression levels more accurately, but also detects trace amounts of gene expression, and its advantages are comprehensive and multi-level. However, many precious human tissue samples are preserved in the form of frozen tissue, and the living cells obtained after digestion by enzymatic hydrolysis are very few, which...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12N15/10
CPCC12N15/1017C12Q1/6806C12Q2527/125C12Q2527/127C12Q2523/32
Inventor 訾晓渊尤园园朱文奇丁修恒
Owner SINGLERON NANJING BIOTECHNOLOGIES LTD
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